Cytopathic adenoviral E1B mutated viruses for therapy and prophylaxis of neoplasia

ABSTRACT

Methods and compositions for treating neoplastic conditions by viral-based therapy are provided. Mutant virus lacking viral proteins which bind and/or inactivate p53 or RB are administered to a patient having a neoplasm which comprises cells lacking p53 and/or RB function. The mutant virus is able to substantially produce a replication phenotype in neoplastic cells but is substantially unable to produce a replication phenotype in non-replicating, non-neoplastic cells having essentially normal p53 and/or RB function. The preferential generation of replication phenotype in neoplastic cells results in a preferential killing of the neoplastic cells, either directly or by expression of a cytotoxic gene in cells expressing a viral replication phenotype.

This application claims priority from U.S. Provisional Application Ser.No. 60/034,615, filed Dec. 31, 1996.

TECHNICAL FIELD

The invention provides compositions of recombinant cytopathic viruseswhich are capable of replication and/or expression of late region genesin neoplastic mammalian cells but are essentially non-replicable innon-neoplastic cells, methods for constructing and propagating suchrecombinant viruses, methods for treating neoplastic disease with suchrecombinant viruses, and therapeutic compositions comprising suchrecombinant viruses.

BACKGROUND

The proliferation of normal cells is thought to be regulated bygrowth-promoting proto-oncogenes counterbalanced by growth-constrainingtumor-suppressor genes. Mutations that potentiate the activities ofproto-oncogenes create the oncogenes that force the growth of neoplasticcells. Conversely, genetic lesions that inactivate tumor suppressorgenes, generally through mutation(s) that lead to a cell beinghomozygous for the inactivated tumor suppressor allele, can liberate thecell from the normal replicative constraints imposed by these genes.Usually, an inactivated tumor suppressor gene (e.g., p53, RB, DCC, NF-1)in combination with the formation of an activated oncogene (i.e., aproto-oncogene containing an activating structural or regulatorymutation) can yield a neoplastic cell capable of essentiallyunconstrained growth (i.e., a transformed cell).

Oncogenic transformation of cells leads to a number of changes incellular metabolism, physiology, and morphology. One characteristicalteration of oncogenically transformed cells is a loss ofresponsiveness to constraints on cell proliferation and differentiationnormally imposed by the appropriate expression of cell-growth regulatorygenes.

While different types of genetic alterations may all lead to alteredexpression or function of cell-growth regulatory genes and to abnormalgrowth, it is generally believed that more than one event is required tolead to neoplastic transformation of a normal cell to a malignant one(Land et al. (1983) Nature 304: 596; Weinberg RA (1989) Cancer Res. 49:3713). The precise molecular pathways and secondary changes leading tomalignant transformation for most cell types are not clear. A number ofcases have been reported in which altered expression or activity of someproteins with putative cell-cycle control functions and/or implicated inthe formation of functional transcriptional complexes, such as p53 andRB, can lead to loss of proliferation control in cells (Ullrich et al.(1992) J. Biol. Chem. 267: 15259; Hollstein et al. (1991) Science 253:49; Sager R (1992) Curr. Opin. Cell. Biol. 4: 155; Levine et al. (1991)Nature 351: 453).

Some oncogenes have been found to possess characteristic activatingmutations in a significant fraction of certain cancers. For example,particular mutations in the ras^(H) and ras^(K) coding regions (e.g.,codon 12, codon 61; Parada et al. (1984) Nature 312: 649) and the APCgene (Powell et al. (1992) Nature 359: 235) are associated withoncogenic transformation of cultured cells and are present in a strikingpercentage of specific human cancers (e.g., colon adenocarcinoma,bladder carcinoma, lung carcinoma and adenocarcinoma, hepatocarcinoma).These findings have led to the development of diagnostic and therapeuticreagents (e.g., polynucleotide probes and antibodies) that specificallyrecognize the activated form(s) of such oncogenes (U.S. Pat. No.4,798,787 and U.S. Pat. No. 4,762,706).

The excessive or inappropriate expression of other oncogenes, such asmyc, erbB-2, and pim-1, appears to be able to potentiate oncogenictransformation without necessarily requiring the presence of activatingmutation(s) in the coding region. Overexpression of erbB-2 is frequentlyfound in adenocarcinoma of the breast, stomach, and ovary, and erbB-2levels in these cell types might serve as a diagnostic marker forneoplasia and/or may correlate with a specific tumor phenotype (e.g.,resistance to specific drugs, growth rate, differentiation state).

Transgenic animals harboring various oncogenes (U.S. Pat. No. 4,736,866and U.S. Pat. No. 5,087,571) or functionally disrupted tumor suppressorgenes (Donehower et al. (1992) Nature 356: 215) have been described foruse in carcinogen screening assays, among other potential uses.

Despite this progress in developing a more defined model of themolecular mechanisms underlying the transformed phenotype and neoplasia,few significant therapeutic methods applicable to treating cancer beyondconventional chemotherapy have resulted. Many conventionalchemotherapeutic agents have a low therapeutic index, with therapeuticdosage levels being at or near dosage levels which produce toxicity.Toxic side effects of most conventional chemotherapeutic agents areunpleasant and lead to life-threatening bone marrow suppression, amongother side effects.

Recent approaches for performing gene therapy to correct or supplementdefective alleles which cause congenital diseases, such as cysticfibrosis, have been attempted with reports of limited initial success.Some gene therapy approaches involve transducing a polynucleotidesequence capable of expressing a functional copy of a defective alleleinto a cell in vivo using replication-deficient recombinant adenovirus(Rosenfeld et al. (1992) Cell 68: 143). Some of these gene therapymethods are efficient at transducing polynucleotides into isolated cellsexplanted from a patient, but have not been shown to be highly efficientin vivo. Therapeutic approaches to cancer which rely on transfection ofexplanted tumor cells with polynucleotides encoding tumor necrosisfactor (TNF) and interleukin-2 (IL-2) have been described (Pardoll D(1992) Curr. Opin. Oncol. 4: 1124).

Although it might someday prove possible for gene therapy methods to beadapted to correct defective alleles of oncogenes or tumor suppressorgenes in transformed cells in vivo, present gene therapy methods havenot been reported to be able to efficiently transduce and correctlytarget (e.g., by homologous recombination) a sufficient percentage ofneoplastic cells for practical gene therapy of neoplasia in situ. Thenature of cancer biology mandates that a substantial fraction of theneoplastic cells, preferably all of the clonal progeny of thetransformed cell, are ablated for an effective therapeutic effect.Moreover, present methods for gene therapy are very expensive, requiringex vivo culturing of explanted cells prior to reintroduction into apatient. Widespread application of such methods, even if they wereeffective, would be prohibitively expensive.

Thus there exists a need in the art for methods and compositions fordiagnosis and therapy of neoplastic diseases, especially for methodswhich selectively ablate neoplastic cells without the undesirablekilling of non-neoplastic cells that is typical of conventionalantineoplastic chemotherapy. The present invention fulfills these andother needs.

The references discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

The present invention provides several novel methods and compositionsfor ablating neoplastic cells by infecting the neoplastic cells with arecombinant adenovirus which is substantially replication deficient innon-neoplastic cells and which exhibits at least a partial replicationphenotype in neoplastic cells. The difference in replication phenotypeof the adenovirus constructs of the invention in neoplastic andnon-neoplastic cells provides a biological basis for viral-based therapyof cancer. Expression of adenoviral cytopathic effects, and optionallyexpression of a negative-selectable drug gene (e.g., HSV tk), arecorrelated with the adenoviral replication phenotype characteristic ofneoplastic cells infected with the recombinant adenovirus constructs ofthe invention, thus discriminating between neoplastic and non-neoplasticcells and providing selective cytotoxicity of neoplastic cells. Althoughthe methods are described in detail specifically for adenoviralconstructs, the methods are believed to be applicable to essentially anyvirus type wherein efficient replication requires binding and/orsequestration and/or inactivation of a host cell protein that is presentin non-neoplastic cells but is substantially absent or nonfunctional inneoplastic cells (e.g., p53, RB).

In order for adenovirus to replicate efficiently in cells, theadenoviral E1b gene product, p55, forms a complex with the host cell p53protein, thereby sequestering and/or inactivating p53 and producing acell that is deficient in p53 function. Such a cell made deficient inp53 function can support replication of the adenovirus. In this way,wild-type adenovirus is able to replicate in cells containing p53, asthe adenovirus p55 proteins inactivates and/or sequesters the host cellp53 protein. In one embodiment of the invention, a recombinantadenovirus comprising an E1b locus encoding a mutant p55 protein that issubstantially incapable of forming a functional complex with p5³ proteinin infected cells is administered to an individual or cell populationcomprising a neoplastic cell capable of being infected by therecombinant adenovirus. The substantial incapacity of the recombinantadenovirus to effectively sequester p53 protein in infectednon-neoplastic cells results in the introduced recombinant adenoviralpolynucleotide(s) failing to express a replication phenotype innon-neoplastic cells. By contrast, neoplastic cells which lack afunctional p53 protein support expression of a replication phenotype bythe introduced recombinant adenovirus which leads to ablation of theneoplastic cell by an adenoviral cytopathic effect and/or expression ofa negative selection gene linked to the replication phenotype. Inpreferred variations of these embodiments, the recombinant adenoviruscomprises an E1b locus encoding a mutant p55 which is substantiallyincapable of binding p53 and may optionally also lack a functional p19protein (i.e., incapable of inhibiting expression of adenoviral earlyregion genes in the presence of E1a polypeptides). Recombinantadenoviruses of the invention may further comprise a mutant p19 genewhich produces enhanced cytopathic effects; such a mutant known in theart is the p19 cyt mutant gene. However, it may be preferable to retainfunctional p19 in some mutants to maintain the integrity of viral DNAduring the infection.

In an alternative embodiment of the invention, a recombinant adenoviruscomprising an E1a locus encoding an E1a protein (e.g., p289R or p243R)that is substantially incapable of forming a complex with RB protein ininfected cells is administered to an individual or cell populationcomprising a neoplastic cell capable of being infected by therecombinant adenovirus. The substantial incapacity of the recombinantadenovirus to effectively sequester RB protein in infectednon-neoplastic cells results in the introduced recombinant adenoviralpolynucleotide(s) failing to express a replication phenotype innon-neoplastic cells. By contrast, neoplastic cells which lack afunctional RB protein support expression of a replication phenotype bythe introduced recombinant adenovirus which leads to ablation of theneoplastic cell by an adenoviral cytopathic effect and/or expression ofa negative selection gene linked to the replication phenotype. Inpreferred variations of these embodiments, the recombinant adenoviruscomprises an E1a locus encoding a mutant E1a protein (e.g., p289R) thatlacks a CR1 and/or CR2 domain capable of binding RB (and/or the 300kDpolypeptide and/or the 107kD polypeptide) but comprises a functional CR3domain capable of transactivation of adenoviral early genes. Additionalvariations of these embodiments include those where the recombinantadenovirus comprises a nonfunctional E1a locus which is substantiallyincapable of expressing a protein that binds to and inactivates RB andmay optionally also comprise a functional p19 protein (i.e., capable ofstimulating expression of adenoviral early region genes in the absenceof E1a function). Recombinant adenoviruses of the invention may furthercomprise a mutant p19 gene which produces enhanced cytopathic effects;such a mutant known in the art is the p19 cyt mutant gene.

The invention provides novel recombinant adenovirus constructs which arereplication defective in non-neoplastic cells but capable of expressinga replication phenotype in neoplastic cells lacking functional p53and/or RB. The novel recombinant adenovirus constructs comprise amutation, such as a deletion or point mutation, in the E1a and/or E1bgene regions, especially in the sequences encoding the E1b p55 proteinand the CR1 and CR2 domains of the E1a p289R or p243R proteins. In someembodiments, a negative selectable gene, such as an HSV tk gene, isoperably linked to an early region (e.g., E2, E1a , E1b)enhancer/promoter, a late region gene enhancer/promoter (e.g., majorlate promoter), or an early or late region promoter with a CMV enhancer,in a recombinant adenovirus construct also comprising an E1a or E1bmutation, so that the negative selectable gene is preferentiallytranscribed in infected cells which express a replication phenotype(i.e., neoplastic cells) and provides negative selection of such cellsby administration of an effective dosage of a negative selection agent(e.g., gancyclovir, FIAU). A negative selectable gene may be inserted inplace of an E1a and/or E1b structural sequence to concomitantly form anE1a.sup.(-) replication deficient mutant, E1b.sup.(-) replicationdeficient mutant, or E1a/E1b double mutant, respectively.

Antineoplastic compositions comprising such recombinant adenovirus in apharmaceutically acceptable form for delivery to a neoplastic cellpopulation in vivo are also provided.

The invention also provides recombinant papovaviruses, such as humanpapillomavirus (HPV), polyomaviruses (e.g., BK, JC) and SV40, which lackfunctional proteins for binding and/or inactivating p53 and/or RB. Humanpapillomavirus mutants lacking expression of functional E6 protein willsubstantially lack the capacity to effectively degrade p53 and thus willbe capable of manifesting a replication phenotype in p53.sup.(-) cellsbut not in cells containing a sufficient level of functional p53. Humanpapillomavirus mutants lacking expression of functional E7 protein willsubstantially lack the capacity to effectively bind RB and thus will becapable of manifesting a replication phenotype in RB.sup.(-) cells butnot in cells containing a sufficient level of functional RB. Humanpapillomavirus mutants lacking expression of both functional E6 proteinand functional E7 protein will substantially lack the capacity toeffectively bind RB and p53 thus will be capable of manifesting areplication phenotype in p53.sup.(-) RB⁻) cells but not in cellscontaining a sufficient level of functional RB and/or p53.

The invention also provides novel methods for treating a neoplasticdisease comprising the steps of administering to a patient a recombinantvirus capable of preferentially expressing a replication phenotypeand/or expressing a cytopathic effect in a neoplastic cell population ascompared to expression in a non-neoplastic cell population.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a schematic representation of the domain structure andprotein-protein interactions of an adenovirus E1a -289R polypeptide.

FIG. 2 shows in schematic form the construction of the plasmids p019,p020, and p021.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The term "naturally-occurring" as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring. As used herein, the term "recombinant" indicatesthat a polynucleotide construct (e.g., and adenovirus genome) has beengenerated, in part, by intentional modification by man.

As used herein, the term "replication deficient virus" refers to a virusthat preferentially inhibits cell proliferation or induces apoptosis ina predetermined cell population (e.g., cells substantially lacking p53and/or RB function) which supports expression of a virus replicationphenotype, and which is substantially unable to inhibit cellproliferation, induce apoptosis, or express a replication phenotype incells comprising normal p53 and RB function levels characteristic ofnon-replicating, non-transformed cells. Typically, a replicationdeficient virus exhibits a substantial decrease in plaquing efficiencyon cells comprising normal RB and/or p53 function.

As used herein, the term "p53 function" refers to the property of havingan essentially normal level of a polypeptide encoded by the p53 gene(i.e., relative to non-neoplastic cells of the same histological type),wherein the p53 polypeptide is capable of binding an E1b p55 protein ofwild-type adenovirus 2 or 5. For example, p53 function may be lost byproduction of an inactive (i.e., mutant) form of p53 or by a substantialdecrease or total loss of expression of p53 polypeptide(s). Also, p53function may be substantially absent in neoplastic cells which comprisep53 alleles encoding wild-type p53 protein; for example, a geneticalteration outside of the p53 locus, such as a mutation that results inaberrant subcellular processing or localization of p53 (e.g., a mutationresulting in localization of p53 predominantly in the cytoplasm ratherthan the nucleus) can result in a loss of p53 function.

As used herein, the term "RB function" refers to the property of havingan essentially normal level of a polypeptide encoded by the RB gene(i.e., relative to non-neoplastic cells of the same histological type),wherein the RB polypeptide is capable of binding an E1a protein ofwild-type adenovirus 2 or 5. For example, RB function may be lost byproduction of an inactive (i.e., mutant) form of RB or by a substantialdecrease or total loss of expression of RB polypeptide(s). Also, RBfunction may be substantially absent in neoplastic cells that compriseRB alleles encoding a wild-type RB protein; for example, a geneticalteration outside of the RB locus, such as a mutation that results inaberrant subcellular processing or localization of RB, may result in aloss of RB function.

As used herein, the term "replication phenotype" refers to one or moreof the following phenotypic characteristics of cells infected with avirus such as a replication deficient adenovirus: (1) substantialexpression of late gene products, such as capsid proteins (e.g.,adenoviral penton base polypeptide) or RNA transcripts initiated fromviral late gene promoter(s), (2) replication of viral genomes orformation of replicative intermediates, (3) assembly of viral capsids orpackaged virion particles, (4) appearance of cytopathic effect (CPE) inthe infected cell, (5) completion of a viral lytic cycle, and (6) otherphenotypic alterations which are typically contingent upon abrogation ofp53 or RB function in non-neoplastic cells infected with a wild-typereplication competent DNA virus encoding functional oncoprotein(s). Areplication phenotype comprises at least one of the listed phenotypiccharacteristics, preferably more than one of the phenotypiccharacteristics.

The term "antineoplastic replication deficient virus" is used herein torefer to a recombinant virus which has the functional property ofinhibiting development or progression of a neoplasm in a human, bypreferential cell killing of infected neoplastic cells relative toinfected non-replicating, non-neoplastic cells of the same histologicalcell type.

As used herein, "neoplastic cells" and "neoplasia" refer to cells whichexhibit relatively autonomous growth, so that they exhibit an aberrantgrowth phenotype characterized by a significant loss of control of cellproliferation. Neoplastic cells comprise cells which may be activelyreplicating or in a temporary non-replicative resting state (G₁ or G₀);similarly, neoplastic cells may comprise cells which have awell-differentiated phenotype, a poorly-differentiated phenotype, or amixture of both type of cells. Thus, not all neoplastic cells arenecessarily replicating cells at a given timepoint. The set defined asneoplastic cells consists of cells in benign neoplasms and cells inmalignant (or frank) neoplasms. Frankly neoplastic cells are frequentlyreferred to as cancer, typically termed carcinoma if originating fromcells of endodermal or ectodermal histological origin, or sarcoma iforiginating from cell types derived from mesoderm.

As used herein, the term "operably linked" refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is"operably linked" when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame. However, sinceenhancers generally function when separated from the promoter by severalkilobases and intronic sequences may be of variable lengths, somepolynucleotide elements may be operably linked but not contiguous.

As used herein, "physiological conditions" refers to an aqueousenvironment having an ionic strength, pH, and temperature substantiallysimilar to conditions in an intact mammalian cell or in a tissue spaceor organ of a living mammal. Typically, physiological conditionscomprise an aqueous solution having about 150 mM NaCl (or optionallyKCl), pH 6.5-8.1, and a temperature of approximately 20-45° C.Generally, physiological conditions are suitable binding conditions forintermolecular association of biological macromolecules. For example,physiological conditions of 150 mM NaCl, pH 7.4, at 37° C. are generallysuitable.

DETAILED DESCRIPTION

Generally, the nomenclature used hereafter and the laboratory proceduresin cell culture, molecular genetics, and molecular virology describedbelow are those well known and commonly employed in the art. Standardtechniques are used for recombinant nucleic acid methods, polynucleotidesynthesis, polypeptide synthesis, generation and propagation of virusstocks (including cell lines capable of trans-complementation ofreplication deficient virus stocks), cell culture, and the like.Generally enzymatic reactions and purification steps are performedaccording to the manufacturer's specifications. The techniques andprocedures are generally performed according to conventional methods inthe art and various general references (see, generally, Sambrook et al.Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Virology, Second edition,eds. Fields BN and Knipe DM, (1990) Raven Press, New York, N.Y.,incorporated herein by reference) which are provided throughout thisdocument. The procedures therein are believed to be well known in theart and are provided for the convenience of the reader. All theinformation contained therein is incorporated herein by reference.

Neoplasia is a pathological condition which is characterized, in part,by the generation of neoplastic cells having variant genotypes andphenotypes. Some tumors may comprise a population of cells lacking RBfunction but having p53 function; such cells are designated RB.sup.(-).Some tumor cells may lack p53 function but have RB function; such cellsare designated p53.sup.(-). Some tumors may comprise cells lacking bothp53 and RB and are designated p53.sup.(-) RB.sup.(-). The cell lineSAOS2 (infra, see Experimental Example) is an example of a neoplasticcell type which is p53.sup.(-) RB.sup.(-). Also, there may be neoplasticcells which comprise essentially normal levels of p53 and RB; such cellshaving both normal p53 and normal RB may lack other oncoproteins (e.g.,tumor suppressor gene products other than p53 or RB) which can providethe basis for antineoplastic viral constructs which can preferentiallymanifest a replication phenotype in such neoplastic cells.

A basis of the present invention is that several DNA viruses whichinfect mammalian cells (e.g., adenoviruses; papovaviruses such as BK andJC, SV40, and papillomaviruses such as HPV, and the like) encode viralproteins which are essential for efficient progression through the viralreplication cycle; some of these viral proteins sequester cellularproteins, such as those involved in cell-cycle control and/or formationof transcription complexes, as a necessary condition for efficient viralreplication. In the absence of the viral proteins which bind, sequester,or degrade such cellular proteins as p53 and RB, viral replication issubstantially inhibited. Normal (i.e., non-neoplastic) cells which areinfected with a mutant virus lacking the ability to sequester or degradep53 and/or RB are generally unable to support replication of the mutantvirus, hence such mutant viruses are considered to be replicationdeficient (or replication defective). However, since the sequestrationor degradation of p53 or RB is not necessary for viral replication incells which lack functional p53 or RB (such cells are designatedp53.sup.(-) and RB.sup.(-) respectively) it is possible that replicationdeficient mutant viruses which are defective for p53 and/or RBsequestration or degradation may express a replication phenotype in suchp53.sup.(-) or RB.sup.(-) cells to a greater extent than in cells havingessentially normal p53 and/or RB function. Neoplastic cells frequentlylack p53 function (a p53.sup.(-) cell), RB function (a RB.sup.(-) cell),or both functions (a p53.sup.(-) RB⁻) cell). Hence, some replicationdeficient viral mutants may preferentially exhibit a replicationphenotype in neoplastic cells.

Viral mutants lacking the capacity to express a functional RBinactivating protein (e.g., adenovirus E1a, HPV E7 protein) willmanifest a replication phenotype in RB.sup.(-) cells and RB.sup.(-)p53.sup.(-) cells. Viral mutants lacking the capacity to express afunctional p53 inactivating protein (e.g., adenovirus E1b p55, HPV E6protein) will manifest a replication phenotype in p53.sup.(-) cells andRB.sup.(-) p53.sup.(-) cells. Viral mutants lacking the capacity toexpress both a functional p53 inactivating protein (e.g., adenovirus E1bp55, HPV E6 protein) and a functional RB inactivating protein (e.g.,adenovirus E1a , HPV E7 protein) will manifest a replication phenotypein RB.sup.(-) p3.sup.(-) cells. Cytotoxicity linked to the expression ofa replicative phenotype can therefore be used as a basis forpreferentially killing neoplastic cells having a RB.sup.(-) p53.sup.(-),or RB.sup.(-) p53.sup.(-) phenotype. Although some replicatingnon-neoplastic cells may transiently exhibit a RB.sup.(-) phenotype,p53.sup.(-) phenotype, or RB.sup.(-) p53.sup.(-) phenotype duringprogression through the cell cycle, the viral mutants of the inventionmay be used for preferential, albeit not necessarily completelyselective, killing of neoplastic cells, thus constituting a usefulantineoplastic therapy modality to be used alone or in combination withother modalities of treatment. Deletions (or other inactivatingmutations) in the 37 amino-terminal residues of the HPV E7 polypeptideare preferred HPV mutants for application to RB.sup.(-) cells, sincethese residues are important for RB binding.

Although the methods and compositions presented below are describedspecifically for methods relating to replication deficient adenoviralconstructs, it is believed that the invention can be practiced withother DNA viruses encoding oncoproteins which sequester or enhance thedegradation of p53 protein or RB protein, for example replicationdeficient papillomavirus species (e.g., mutants of HPV types 16, 18, 33)that contain mutations in the E6 and/or E7 genes which substantiallyabrogate p53 and/or RB function, respectively. In addition to members ofthe family Adenoviridae (specifically the genus Mastadenovirus), it isbelieved that members of the family Papovaviridae, especiallypapillomavirus and polyomavirus, which encode viral proteins thatsequester and/or inactivate p53 or RB are suitable for use in themethods of the invention.

For a general description of adenovirus and papovavirus biology,Virology, Second edition, eds. Fields BN and Knipe DM, Vol.2, pp.1651-1740, Raven Press, New York, N.Y., incorporated herein byreference, may be referred to for guidance. The following specificdescriptions refer to, but are not limited to, adenovirus serotype 5 andadenovirus serotype 2. Although it is believed that other adenoviralserotypes may be used, adenovirus type 5 provides a common referencepoint for the nucleotide numbering convention of viral polynucleotidesand amino acid numbering of viral-encoded polypeptides of the E1a viralgene region, and other viral genes. Adenovirus type 2 provides aconvenient reference for the numbering convention of the E1b viral generegion, and other viral gene regions. It is believed that those of skillin the art will readily identify the corresponding positions in otheradenoviral serotypes. References to human papillomavirus generally referto a type associated with neoplasia (e.g., types 16, 18, or 33),although non-oncogenic types may also be used.

E1a Mutants

The loss of retinoblastoma tumor suppressor gene (RB) gene function hasbeen associated with the etiology of various types of tumors. Theproduct of this tumor suppressor gene, a 105 kilodalton polypeptidecalled pRB or p105, is a cell-cycle regulatory protein. The pRBpolypeptide inhibits cell proliferation by arresting cells at the G₁phase of the cell cycle. The pRB protein is also a major target ofseveral DNA virus oncoproteins, including adenovirus E1a, SV40 large TAg, and papillomavirus E7. These viral proteins bind and inactivate pRB,and the function of inactivating pRB is important in facilitating viralreplication. The pRB protein interacts with the E2F transcriptionfactor, which is involved in the expression of the adenovirus E2 geneand several cellular genes, and inhibits the activity of thistranscription factor (Bagchi et al. (1991) Cell 65: 1063; Bandara et al.(1991) Nature 351: 494; Chellappan et al. (1992) Proc. Natl. Acad. Sci.(U.S.A.) 89: 4549, incorporated herein by reference). The viraloncoproteins, such as adenovirus E1a, disrupt the pRB/E2F complexresulting in activation of E2F. However, cells lacking sufficientfunctional pRB to complex the E2F will not require the presence of afunctional oncoprotein, such as E1a, to possess transcriptionally activeE2F. Therefore, it is believed that replication deficient adenovirusspecies which lack the capacity to complex RB but substantially retainother essential replicative functions will exhibit a replicationphenotype in cells which are deficient in RB function (e.g., cells whichare homozygous or heterozygous for substantially deleted RB alleles,cells which comprise RB alleles encoding mutant RB proteins which areessentially nonfunctional, cells which comprise mutations that result ina lack of function of an RB protein) but will not substantially exhibita replicative phenotype in non-replicating, non-neoplastic cells. Suchreplication deficient adenovirus species are referred to herein forconvenience as E1a -RB.sup.(-) replication deficient adenoviruses.

A cell population (such as a mixed cell culture or a human cancerpatient) which comprises a subpopulation of neoplastic cells lacking RBfunction and a subpopulation of non-neoplastic cells which expressessentially normal RB function can be contacted under infectiveconditions (i.e., conditions suitable for adenoviral infection of thecell population, typically physiological conditions) with a compositioncomprising an infectious dosage of a E1a -RB.sup.(-) replicationdeficient adenovirus. Such contacting results in infection of the cellpopulation with the E1a -RB.sup.(-) replication deficient adenovirus.The infection produces preferential expression of a replicationphenotype in a significant fraction of the cells comprising thesubpopulation of neoplastic cells lacking RB function but does notproduce a substantial expression of a replicative phenotype in thesubpopulation of non-neoplastic cells having essentially normal RBfunction. The expression of a replication phenotype in an infectedRB.sup.(-) cell results in the death of the cell, such as by cytopathiceffect (CPE), cell lysis, apoptosis, and the like, resulting in aselective ablation of neoplastic RB.sup.(-) cells from the cellpopulation.

Typically, E1a -RB.sup.(-) replication deficient adenovirus constructssuitable for selective killing of RB(-) neoplastic cells comprisemutations (e.g., deletions, substitutions, frameshifts) which inactivatethe ability of an E1a polypeptide to bind RB protein effectively. Suchinactivating mutations typically occur in the E1a CR1 domain (aminoacids 30-85 in Ad5: nucleotide positions 697-790) and/or the CR2 domain(amino acids 120-139 in Ad5; nucleotide positions 920-967), which areinvolved in binding the p105 RB protein and the p107 protein.Preferably, the CR3 domain (spanning amino acids 150-186) remains and isexpressed as a truncated p289R polypeptide and is functional intransactivation of adenoviral early genes. FIG. 1 portrays schematicallythe domain structure of the E1a-289R polypeptide.

Suitable E1a-RB.sup.(-) replication deficient adenovirus constructs foruse in the methods and compositions of the invention include, but arenot limited to the following examples: (1) adenovirus serotype 5 NT dl1010, which encodes an E1a protein lacking the CR1 and CR2 domains(deletion of amino acids 2 to 150; nucleotides 560-1009) necessary forefficient RB binding, but substantially retaining the CR3 domain (Whyteet al. (1989) Cell 56: 67, incorporated herein by reference), and (2)adenovirus serotype 5 dl 312, which comprises a deleted viral genomelacking the region spanning nucleotides 448-1349 which encodes theentire E1a region in wild-type adenovirus (Jones N and Shenk T (1979)Proc. Natl. Acad. Sci. (U.S.A.) 76: 3665, incorporated herein byreference). Ad5 NT dl 1010 is a preferred E1a-RB.sup.(-) replicationdeficient adenovirus and is available from Dr. E. Harlow, MassachusettsGeneral Hospital, Boston, Mass.).

It may be preferable to incorporate additional mutations into suchadenovirus constructs to inhibit formation of infectious virions inneoplastic cells which otherwise would support replication of theE1a-RB.sup.(-) mutants. Such additional inactivating mutations would bepreferred in therapeutic modalities wherein complete viral replicationforming infectious virions capable of spreading to and infectingadjacent cells is undesirable. These fully inactivated mutants arereferred to as nonreplicable E1a-RB.sup.(-) mutants. Such nonreplicablemutants comprise mutations which prevent formation of infectious virionseven in p53.sup.(-) RB.sup.(-) cells; such mutations typically arestructural mutations in an essential virion protein or protease.

However, in many modalities it is desirable for the mutant virus to bereplicable and to form infectious virions containing the mutant viralgenome which may spread and infect other cells, thus amplifying theantineoplastic action of an initial dosage of mutant virus.

Additional E1a.sup.(-) mutants lacking the capacity to bind RB can begenerated by those of skill in the art by generating mutations in theE1a gene region encoding E1a polypeptides, typically in the CR1 and/orCR2 domains, expressing the mutant E1a polypeptide, contacting themutant E1a polypeptides with p105 or a binding fragment of RB underaqueous binding conditions, and identifying mutant E1a polypeptideswhich do not specifically bind RB as being candidate E1a.sup.(-) mutantssuitable for use in the invention. Alternative assays include contactingthe mutant E1a polypeptides with the 300kD protein and/or p107 proteinor binding fragment thereof under aqueous binding conditions, andidentifying mutant E1a polypeptides which do not specifically bind the300kD and/or p107 polypeptides as being candidate E1a.sup.(-) mutantssuitable for use in the invention. Alternative binding assays includedetermining the inability of E1a.sup.(-) mutant protein (or absence ofE1a protein) to form complexes with the transcription factor E2F and/orto lack the ability to dissociate the RB protein from RB:E2F complexesunder physiological conditions (Chellappan et al. (1991) op.cit.,incorporated herein by reference).

Alternative functional assays for determining mutants lacking E1afunction, such as loss of transctivation by E1a of transcription ofvarious reporter polypeptides linked to a E1a-dependent transcriptionalregulatory sequence, and the like, will be used.

E1b Mutants

A function of the cellular phosphoprotein p53 is to inhibit theprogression of mammalian cells through the cell cycle. Wild-typeadenovirus E1b p55 protein binds to p53 in infected cells that have p53and produce a substantial inactivation of p53 function, likely bysequestering p53 in an inactive form. Functional E1b p55 protein isessential for efficient adenoviral replication in cells containingfunctional p53. Hence, adenovirus variants which substantially lack theability to bind p53 are replication deficient in non-replicating,non-neoplastic cells having normal levels of functional p53.

Human tumor cells frequently are homozygous or heterozygous for mutated(e.g., substitution, deletion, frameshift mutants) p53 alleles, and lackp53 function necessary for normal control of the cell cycle (Hollsteinet al. (1991) Science 253: 49; Levine et al. (1991) op.cit.,incorporated herein by reference). Thus, many neoplastic cells arep53.sup.(-), either because they lack sufficient levels of p53 proteinand/or because they express mutant forms of p53 which are incapable ofsubstantial p53 function, and which may substantially diminish p53function even when wild-type p53 may be present (e.g., by inhibitingformation of functional multimers). Some neoplastic cells may comprisealleles encoding essentially wild-type p53 proteins, but may comprise asecond site mutation that substantially abrogates p53 function, such asa mutation that results in p53 protein being localized in the cytoplasmrather than in the nucleus; such second site mutants also substantiallylack p53 function.

It is believed that replication deficient adenovirus species which lackthe capacity to complex p53 but substantially retain other essentialviral replicative functions will exhibit a replication phenotype incells which are deficient in p53 function (e.g., cells which arehomozygous for substantially deleted p53 alleles, cells which comprisemutant p53 proteins which are essentially nonfunctional) but will notsubstantially exhibit a replicative phenotype in non-replicating,non-neoplastic cells. Such replication deficient adenovirus species arereferred to herein for convenience as E1b-p53.sup.(-) replicationdeficient adenoviruses.

A cell population (such as a mixed cell culture or a human cancerpatient) which comprises a subpopulation of neoplastic cells lacking p53function and a subpopulation of non-neoplastic cells which expressessentially normal p53 function can be contacted under infectiveconditions (i.e., conditions suitable for adenoviral infection of thecell population, typically physiological conditions) with a compositioncomprising an infectious dosage of a E1b-p53.sup.(-) replicationdeficient adenovirus. Such contacting results in infection of the cellpopulation with the E1b-p53.sup.(-) replication deficient adenovirus.The infection produces preferential expression of a replicationphenotype in a significant fraction of the cells comprising thesubpopulation of neoplastic cells lacking p53 function but does notproduce a substantial expression of a replicative phenotype in thesubpopulation of non-neoplastic cells having essentially normal p53function. The expression of a replication phenotype in an infectedp53.sup.(-) cell results in the death of the cell, such as by cytopathiceffect (CPE), cell lysis, apoptosis, and the like, resulting in aselective ablation of neoplastic p53.sup.(-) cells from the cellpopulation.

Typically, E1b-p53.sup.(-) replication deficient adenovirus constructssuitable for selective killing of p53.sup.(-) neoplastic cells comprisemutations (e.g., deletions, substitutions, frameshifts) which inactivatethe ability of the E1b p55 polypeptide to bind p53 protein effectively.Such inactivating mutations typically occur in the regions of p55 whichbind p53. Optionally, the mutant E1b region may encode and express afunctional p19 protein encoded by the E1b region remains and that isfunctional in transactivation of adenoviral early genes in the absenceof E1a polypeptides.

Suitable E1b-p53.sup.(-) replication deficient adenovirus constructs foruse in the methods and compositions of the invention include, but arenot limited to the following examples: (1) adenovirus type 2 dl 1520,which contains a C to T mutation at nucleotide position 2022 thatgenerates a stop codon 3 amino acids downstream of the AUG codon usedfor initiation of translation of the p55 protein and a deletion betweennucleotides 2496 and 3323 replaced with a small linker insertion thatgenerates a second stop codon at nucleotide 3336; the expression of thep19 protein is essentially unaffected (Barker and Berk (1987) Virology156: 107, incorporated herein by reference, and (2) a compositeadenovirus construct comprising adenovirus type 2 dl 1520 comprising atleast the position 2022 mutation and/or the 2496-3323 deletion mutation,or a substantial portion thereof, and an additional mutation in p19 toyield a p19 cyt mutant; the composite virus construct lacks p55 andcomprises the enhanced cytopathic effect of the p19 cyt mutation. Ad2 dl1520 are available from Dr. A. Berk, University of California at LosAngeles, Los Angeles, Calif., and are described in the literature,including Barker and Berk (1987) Virology 156: 107, incorporated hereinby reference.

It may be preferable to incorporate additional mutations into suchadenovirus constructs to inhibit formation of infectious virions inneoplastic cells which otherwise would support replication of theE1b-p53.sup.(-) mutants. Such additional inactivating mutations would bepreferred in therapeutic modalities wherein complete viral replicationforming infectious virions capable of spreading to and infectingadjacent cells is undesirable. These fully inactivated mutants arereferred to as nonreplicable E1b-p53.sup.(-) mutants. Such nonreplicablemutants comprise mutations which prevent formation of infectious virionseven in p53.sup.(-) RB.sup.(-) cells; such mutations typically arcstructural mutations in an essential virion protein or protease.

However, in many modalities it is desirable for the mutant virus to bereplicable and to form infectious virions containing the mutant viralgenome which may spread and infect other cells, thus amplifying theantineoplastic action of an initial dosage of mutant virus.

Additional E1b.sup.(-) mutants lacking the capacity to bind p53 can begenerated by those of skill in the art by generating mutations in theE1b gene region encoding the p55 polypeptide, expressing mutant p55polypeptides, contacting the mutant p55 polypeptides with p53 or abinding fragment of p53 under aqueous binding conditions, andidentifying mutant E1b polypeptides which do not specifically bind p53as being candidate E1b.sup.(-) mutants suitable for use in theinvention.

More typically, a functional assay will be used to identify candidateE1b.sup.(-) mutants. For example, the Friend assay for determination ofp53 function will be performed essentially as described in Frebourg etal. (1992) Cancer Res. 52: 6977, incorporated herein by reference. E1bmutants which lack the capacity to inactivate p53 will be identified ascandidate E1b.sup.(-) replication deficient mutants.

E1a/E1b Double Mutants

Some human tumor cells lack both p53 function and RB function, either bymutational inactivation or deletion of one or both protein species. Suchcells are termed p53.sup.(-) RB.sup.(-) cells.

It is believed that replication deficient adenovirus species which lackthe capacity to bind p53 and which also lack the capacity to bind RB,but which substantially retain other essential viral replicativefunctions will preferentially exhibit a replication phenotype inp53.sup.(-) RB.sup.(-) cells. Such replication deficient adenovirusspecies are referred to herein for convenience as E1a-RB.sup.(-)/E1b-p53.sup.(-) replication deficient adenoviruses, or simply E1a/E1bdouble mutants. Such E1a/E1b double mutants can be constructed by thoseof skill in the art by combining at least one E1a-RB.sup.(-) mutation inthe E1a region and at least one E1b-p53.sup.(-) mutation in E1b regionencoding p55 to form a E1a/E1b double mutant adenovirus. Such areplication deficient double mutant adenovirus will exhibit areplication phenotype in cells which are deficient in both p53 and RBfunctions but will not substantially exhibit a replicative phenotype innon-replicating, non-transformed cells or in cells which are deficientin either p53 or RB function but not both functions. For example, theAd5 dl 434 mutant (Grodzicker et al. (1980) Cell 21: 454, incorporatedherein by reference) comprises a deletion of the E1a locus and a partialdeletion of the E1b locus, and substantially lacks the capacity toencode functional E1a and E1b p55 proteins.

A cell population (such as a mixed cell culture or a human cancerpatient) which comprises a subpopulation of neoplastic cells lacking p53and RB functions and a subpopulation of non-neoplastic cells whichexpress essentially normal p53 function and/or RB function can becontacted under infective conditions (i.eg., conditions suitable foradenoviral infection of the cell population, typically physiologicalconditions) with a composition comprising an infectious dosage of areplication deficient E1a/E1b double mutant adenovirus. Such contactingresults in infection of the cell population with the E1a/E1b doublemutant replication deficient adenovirus. The infection producespreferential expression of a replication phenotype in a significantfraction of the cells comprising the subpopulation of neoplastic cellslacking both p53 function and RB function but does not produce asubstantial expression of a replicative phenotype in the subpopulationof non-neoplastic cells having essentially normal p53 function and/or RBfunction. The expression of a replication phenotype in an infectedp53.sup.(-) RB.sup.(-) cell results in the death of the cell, such as bycytopathic effect (CPE), cell lysis, and p the like, resulting in aselective ablation of neoplastic p53.sup.(-) RB.sup.(-) cells from thecell population.

It may be preferable to incorporate additional mutations into suchadenovirus constructs to inhibit formation of infectious virions inneoplastic cells which otherwise would support replication of an E1a/E1bdouble mutant. Such additional inactivating mutations would be preferredin therapeutic modalities wherein complete viral replication forminginfectious virions capable of spreading to and infecting adjacent cellsis undesirable. These fully inactivated mutants are referred to asnonreplicable E1a/E1b double mutants. Such nonreplicable mutantscomprise mutations which prevent formation of infectious virions even inp53.sup.(-) RB.sup.(-) cells; such mutations typically are structuralmutations in an essential virion protein or protease.

However, in many modalities it is desirable for the mutant virus to bereplicable and to form infectious virions containing the mutant viralgenome which may spread and infect other cells, thus amplifying theantineoplastic action of an initial dosage of mutant virus.

Negative Selection Viral Constructs

Although expression of an adenoviral replication phenotype in aninfected cell correlates with viral-induced cytotoxicity, generally bycell lysis, cytopathic effect (CPE), apoptosis, or other mechanisms ofcell death, it may often be preferable to augment the cytotoxicity of arecombinant adenovirus that is to be used for antineoplastic therapy.Such augmentation may take the form of including a negative selectiongene in the recombinant adenovirus, typically operably linked to anadenoviral promoter which exhibits positive transcriptional modulationin cells expressing a replication phenotype. For example, a HSV tk genecassette may be operably linked immediately downstream of an E3 promoterof a replication deficient adenovirus, such as Ad5 NT dl 1110.Frequently, it is desirable to delete a nonessential portion (i.eg., forviral replication and packaging) of the adenoviral genome to accommodatethe negative selection cassette; thus a substantial portion of the E3gene region may be deleted and replaced with a negative selectioncassette such as an HSV tk gene operably linked to an E2 promoter (andenhancer) or other suitable promoter/enhancer. Alternatively, a negativeselection gene may be operably linked to an adenovirus late regionpromoter to afford efficient expression of the negative selection geneproduct in cells expressing a replication phenotype characterized bytranscription from late gene promoters.

For embodiments where viral replication forming infectious virions invivo is undesirable, adenovirus replication deficient constructs whichare nonreplicable are used. Such nonreplicable mutants comprise anE1a.sup.(-) and/or E1b.sup.(-) mutation and comprise all geneticfunctions necessary for generating a replication phenotype in a suitableneoplastic cell (e.g., a p53.sup.(-) cell, a RB.sup.(-) cell, or ap53.sup.(-) RB.sup.(-) cell) but have deleted at least one essentialgene function necessary for formation of infectious virions, such asstructural coat proteins, proteases, and the like. Alternatively, anelicited immune response evoked by the virus may neutralize infectiousvirions and moderate spread of the viral infection. Nonreplicablemutants lacking a complementable trans-acting function in addition to anE1a and/or E1b mutation may be propagated in conjunction with acomplementary helper virus or a helper cell line capable of providingthe deleted trans-acting function(s). For example, the 293 cell line(ATCC # CRL 1573; Graham et al. (1977) J. Gen. Virol. 36: 59,incorporated herein by reference) which provides E1a and E1b functionsin trans may be modified to provide additional functions in trans, suchas a virion coat protein or the like, to permit propagation of the"nonreplicable" mutants for developing virus stocks.

Expression of the HSV tk gene in a cell is not directly toxic to thecell, unless the cell is exposed to a negative selection agent such asgancyclovir or FIAU. Infected cells expressing a replication phenotypewherein a negative selection gene is substantially expressed may produceessentially no additional cytotoxicity until the negative selectionagent (e.g., gancyclovir) is administered in an effective selectivedosage, at which time the infected cells expressing the tk gene will beselectively ablated; thus negative selection can be used for enhancedcytopathic killing and/or to damp out further viral replication bykilling cells exhibiting a replicative phenotype. Further, by adjustingthe dosages and/or administration schedule of the negative selectionagent, it is possible to produce only a partial ablation of the infectedcell population expressing the negative selection gene. Generally, thedosage of gancyclovir is calibrated by generating a standarddose-response curve and determining the dosage level at which a desiredlevel of ablation of infected neoplastic cells is observed. Informationregarding administration of gancyclovir (GANC) to animals is availablein various sources in the art, including human prescribing directionsfrom package inserts. When used in cell culture, a selectiveconcentration of gancyclovir is typically in the range of 0.05mM to 50mM, typically about 1 mM, with about 0.2 mM used for in vitroapplications and about 1-5 mM administered for in vivo applications(typically administered over about 24 hours by continuous infusion froman osmotic pump loaded with 125 mg/ml of gancyclovir in aqueoussolution). A dosage schedule for in vivo administration may comprisegancyclovir at a dosage of 5 mg/kg bodyweight B.I.D., givenintravenously for seven days.

Negative selection genes may be incorporated into E1a-RB.sup.(-)replication deficient adenovirus constructs, E1ba-p53.sup.(-)replication deficient adenoviral constructs, E1a/E1b double mutantreplication deficient viral constructs, or the like. A preferredembodiment is an HSV tk gene cassette (Zjilstra et al. (1989) Nature342:435; Mansouret al. (1988) Nature 336: 348; Johnson et al. (1989)Science 245: 1234: Adairet al. (1989) Proc. Natl. Acad. Sci (U.S.A.) 86:4574; Capecchi, M. (1989) Science 244:1288, incorporated herein byreference) operably linked to the E2 promoter of Ad5 NT dl1110 or analternative promoter and/or enhancer (e.g., major late promoter, E1apromoter/enhancer, E1b promoter/enhancer), with a polydenylation site toform a tk expression cassette. The tk expression cassette (or othernegative selection expression cassette) is inserted into the adenoviralgenome, for example, as a replacement for a substantial deletion of theE3 gene. Other negative selection genes and replication deficientadenovirus constructs will be apparent to those of skill in the art. Itis believed that a negative selection gene operably linked to the E2promoter is an especially preferred embodiment for incorporation intoE1a.sup.(-) replication-deficient adenovirus mutants, as the E2 promotercontains multiple E2F sites, whereas RB.sup.(-) and p53.sup.(-) RB⁻)lack RB function and presumably will exhibit more efficienttranscription from the E2 promoter.

Diagnostic and In Vitro Uses

The replication deficient adenoviruses of the invention may be used todetect the presence of cells lacking p53 and/or RB function. Forexample, a cell sample comprising a subpopulation of neoplastic cellslacking p53 and/or RB can be infected with a suitable replicationdeficient adenovirus species. After a suitable incubation period, thecells in the cell sample that express a replication phenotype (e.g.,loss of ability to exclude Trypan blue, virion formation, ³ H-thymidineincorporation into viral DNA) can be quantified to provide a measure ofthe number or proportion of replicative and/or neoplastic cells in thecell sample. Such methods may be used to diagnose neoplasms and/orevaluate tumor cell load following chemotherapy in a patient on thebasis of an explanted cell sample (e.g., a lymphocyte sample from apatient undergoing chemotherapy for a lymphocytic leukemia).

Alternative diagnostic uses and variations are apparent; for example, areporter gene (e.g., luciferase, b-galactosidase) may be substituted fora negative selection gene in a replication deficient adenovirus;transformed cells may be scored (such as in a cellular sample ortransformation assay) by the expression of the reporter gene, which iscorrelated with expression of a replication phenotype indicating a lackof p53 and/or RB in a cell.

Therapeutic Methods

Therapy of neoplastic disease may be afforded by administering to apatient a composition comprising replication defective adenoviruses ofthe invention, including: E1a-RB.sup.(-) replication deficientadenoviruses, E1b-p53.sup.(-) replication deficient adenoviruses,E1a/E1b double mutants, and replication deficient adenoviruses furthercomprising a negative selection gene.

Various human neoplasms comprising cells that lack p53 and/or RBfunctions may be treated with the replication deficient adenoviralconstructs. For example but not limitation, a human patient or nonhumanmammal having a bronchogenic carcinoma, nasopharyngeal carcinoma,laryngeal carcinoma, small cell and non-small cell lung carcinoma, lungadenocarcinoma, hepatocarcinoma, pancreatic carcinoma, bladdercarcinoma, colon carcinoma, breast carcinoma, cervical carcinoma,ovarian carcinoma, or lymphocytic leukemias may be treated byadministering an effective antineoplastic dosage of an appropriatereplication deficient adenovirus. Suspensions of infectious adenovirusparticles may be applied to neoplastic tissue by various routes,including intravenous, intraperitoneal, intramuscular, subdermal, andtopical. A adenovirus suspension containing about 10³ to 10¹² or morevirion particles per ml may be inhaled as a mist (e.g., for pulmonarydelivery to treat bronchogenic carcinoma, small-cell lung carcinoma,non-small cell lung carcinoma, lung adenocarcinoma, or laryngeal cancer)or swabbed directly on a tumor site for treating a tumor (e.g.,bronchogenic carcinoma, nasopharyngeal carcinoma, laryngeal carcinoma,cervical carcinoma) or may be administered by infusion (e.g., into theperitoneal cavity for treating ovarian cancer, into the portal vein fortreating hepatocarcinoma or liver metastases from other non-hepaticprimary tumors) or other suitable route, including direct injection intoa tumor mass (e.g., a breast tumor), enema (e.g., colon cancer), orcatheter (e.g., bladder cancer).

Candidate antineoplastic adenovirus mutants may be further evaluated bytheir capacity to reduce tumorigenesis or neoplastic cell burden innu/nu mice harboring a transplant of neoplastic cells lacking p53 and/orRB function, as compared to untreated mice harboring an equivalenttransplant of the neoplastic cells.

Antineoplastic replication deficient adenovirus mutants may beformulated for therapeutic and diagnostic administration to a patienthaving a neoplastic disease.

For therapeutic or prophylactic uses, a sterile composition containing apharmacologically effective dosage of one or more species ofantineoplastic replication deficient adenovirus mutant is administeredto a human patient or veterinary non-human patient for treatment of aneoplastic condition. Generally, the composition will comprise about 10³to 10¹⁵ or more adenovirus particles in an aqueous suspension. Apharmaceutically acceptable carrier or excipient is often employed insuch sterile compositions. A variety of aqueous solutions can be used,eg., water, buffered water, 0.4% saline, 0.3% glycine and the like.These solutions are sterile and generally free of particulate matterother than the desired adenoviral virions. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, etc. Excipients which enhance infection of cells by adenovirusmay be included.

Replication deficient viruses may be delivered to neoplastic cells byliposome or immunoliposome delivery; such delivery may be selectivelytargeted to neoplastic cells on the basis of a cell surface propertypresent on the neoplastic cell population (e.g., the presence of a cellsurface protein which binds an immunoglobulin in an immunoliposome).Typically, an aqueous suspension containing the virions are encapsulatedin liposomes or immunoliposomes. For example, a suspension ofreplication deficient adenovirus virions can be encapsulated in micellesto form immunoliposomes by conventional methods (U.S. Pat. No.5,043,164, U.S. Pat. No. 4,957,735, U.S. Pat. No. 4,925,661; Connor andHuang (1985) J. Cell Biol. 101: 582; Lasic DD (1992) Nature 355: 279;Novel Drug Delivery (eds. Prescott LF and Nimmo WS: Wiley, New York,1989); Reddy et al. (1992) J. Immunol. 148: 1585; incorporated herein byreference). Immunoliposomes comprising an antibody that bindsspecifically to a cancer cell antigen (e.g., CALLA, CEA) present on thecancer cells of the individual may be used to target virions to thosecells.

The compositions containing the present antineoplastic replicationdeficient adenoviruses or cocktails thereof can be administered forprophylactic and/or therapeutic treatments of neoplastic disease. Intherapeutic application, compositions are administered to a patientalready affected by the particular neoplastic disease, in an amountsufficient to cure or at least partially arrest the condition and itscomplications. An amount adequate to accomplish this is defined as a"therapeutically effective dose" or "efficacious dose." Amountseffective for this use will depend upon the severity of the condition,the general state of the patient, and the route of administration.

In prophylactic applications, compositions containing the antineoplasticreplication deficient adenoviruses or cocktails thereof are administeredto a patient not presently in a neoplastic disease state to enhance thepatient's resistance to recurrence of a neoplasm or to prolong remissiontime. Such an amount is defined to be a "prophylactically effectivedose." In this use, the precise amounts again depend upon the patient'sstate of health and general level of immunity.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the antineoplastic replication deficient adenoviruses ofthis invention sufficient to effectively treat the patient.

Antineoplastic replication deficient adenoviral therapy of the presentinvention may be combined with other antineoplastic protocols, such asconventional chemotherapy and/or immunosuppression. The procedures forimmunosuppression would consist of those essentially described in WO96/12406. Generally, a replication deficient adenovirus would beadministered alone or in admixture with an immunosuppressive agent.Examples of preferred immunosuppressive compounds includecyclophosphamide, cyclosporine, or dexamethasone. The dosages of thesecompounds to realize a desired level of immunosuppression are known inthe art. For example, cyclophosphamide is preferrably administered atbetween 100-300 mg/kg, while dexamethasone is preferrably administeredat about 2-6 mg/kg.

Propagation of Mutant Adenovirus

Adenoviral mutants of the invention (e.g., E1a-RB.sup.(-) replicationdeficient adenoviruses, E1b-p53.sup.(-) replication deficientadenoviruses, and E1a/E1b double mutants) typically are propagated asviral stocks in a cell line (e.g., the 293 cell line ATCC # CRL 1573,American Type Culture Collection, Manassas, Va.; Graham et al. (1977) J.Gen. Virol. 36: 59) which can provide E1a function, E1b function, orboth E1a and E1b functions, respectively, in trans to supportreplication and formation of infectious mutant virions.

The following examples are offered by way of example and not by way oflimitation. Variations and alternate embodiments will be apparent tothose of skill in the art.

EXPERIMENTAL EXAMPLES Effect of Replication-Deficient RecombinantAdenovirus on Neoplastic Cells lacking p53 and/or Rb

The following experimental example demonstrates that administering areplication-deficient recombinant adenovirus preparation to neoplasticcells lacking p53 and/or Rb function can effectively kill neoplasticcells. The example also shows that non-neoplastic cells containing p53and Rb function are relatively resistant to killing by thereplication-deficient recombinant adenovirus preparation. Therefore, thedata presented hereinbelow provide experimental verification thatadministration of replication-deficient recombinant adenovirus can beused to selectively kill neoplastic cells. The selective killing isprovided by exploiting the differential ability of the mutantadenoviruses to induce a replication phenotype in neoplastic cells, butsubstantially not induce a replication phenotype (or associatedcytopathic effect) in non-neoplastic cells.

Control non-neoplastic cells and cell lines representing a variety ofneoplastic cell types were plated in 6-well culture dishes at or nearconfluence in DMEM (high glucose) with 10% fetal bovine serum andincubated at 37° C., 5% CO₂ under standard culturing conditions. Cellswere plated to be screened at a density of 5×10⁵ cells/well. Theneoplastic cell lines tested were: SAOS-2 (ATCC HTB85), derived from ahuman primary osteogenic sarcoma; U-2OS (ATCC HTB96), derived from ahuman osteogenic sarcoma; HS700T, (ATCC HTB 147), derived from a humanmetastatic adenocarcinoma originating in the pancreas or intestines; 293(ATCC CRL1573), transformed human embryonal kidney; and DLD-1 (ATCCCCL221), derived from a human colon adenocarcinoma. Each of the celllines is available from American Type Culture Collection, Manassas, Va.The control non-neoplastic cells were IMR90 (ATCC CCL 186) and WI-38(ATCC CCL 75), both diploid human lung fibroblast lines.

These cell cultures were subsequently infected, in parallel, withwild-type adenovirus Type 2, and replication-deficient recombinantadenovirus mutants dl 1010, dl 434, and dl 1520. An extra culture dishwas plated for the purpose of counting cells. These cells came from thesame suspension of cells used for the viral infections. Cells werecounted in order to determine the number of viral plaque-forming units(PFU) to add to the cell cultures for a desired multiplicity ofinfection (MOI). The wild-type adenovirus Type 2 and the mutantadenoviruses were added to the parallel cell cultures at MOIs of 0.1,1.0, 10, and 100. Virus suspended in PBS was added to the cell wells ina volume of 1 ml. The inoculated culture dishes were rocked in both theX- and Y- axes immediately after inoculation and halfway through theadsorption time of approximately one hour at 37° C., 5% CO₂. After theone hour adsorption period, 2 ml of DMEM with high glucose and 2% fetalbovine serum was added, and the cultures incubated at 37° C., 5% CO₂under standard culturing conditions. At various times alter infectioncell cultures were stained with 0.5% crystal violet in 20% methanol inorder to determine the efficacy of cell killing by the viruspreparations. Dead cells were detached and rinsed out of the wells,whereas living cells remained in the well and were stained with the dye.The results demonstrate that the replication-deficient recombinantadenovirus preparations were able to preferentially kill neoplasticcells as compared to non-neoplastic cells, and that wild-type adenovirusType 2 killed both neoplastic and non-neoplastic cells approximatelyequally well. More particularly, the results demonstrated that the dl1010 mutant was particularly effective at killing neoplastic cells, withthe dl 1520 mutant and the dl 434 mutant also being effective. Wild-typeadenovirus 2, over days 4-7 post-infection, was able to kill cells ineach of the culture wells, although with variable efficacy andincompletely, with at least some cells staining in each well. Note thatWI-38 and SAOS-2 lines are not good hosts for adenovirus infection and,as discussed below, IMR90, serves as an alternate control cell line forhuman diploid fibroblasts. The dl 1010 mutant, over days 8-12 was ableto kill effectively all of the neoplastic cell lines except theinfection-resistant SAOS-2. Dl 1010 killed the 293, U2OS, HS700T, andDLD-1 lines by 12 days after infection and did not substantially killdiploid human lung fibroblasts (WI-38). The dl 1520 mutant was able tokill effectively 3 of the 5 neoplastic cell lines (293, HS700T, andDLD-1) by 14-20 days after infection and did not substantially killdiploid human lung fibroblasts (WI-38). dl 1520 is an EIB.sup.(-)mutant, and cell line U2OS does not allow such an E1B.sup.(-) mutantvirus to replicate, indicating specificity of the different transformedcell lines for infection by the mutant recombination-defectiveadenoviruses, as predicted. The dl 434 mutant (a double-mutant:E1A.sup.(-) E1B.sup.(-)) was able to kill effectively 3 of the 5neoplastic cell lines (293, HS700T, and DLD-1) by 14-20 days afterinfection and did not substantially kill diploid human lung fibroblasts(WI-38). The DLD-1 and U2OS lines displayed a partially resistantphenotype for replication of dl 434. Each cell line was mock infectedwith PBS alone as a control for viability.

The following experiments were also conducted using IMR90 cells (ATCCCCL 186) and wild-type and mutant adenoviruses. IMR90 cells wereinfected with the indicated virus at MOIs corresponding to 0.1, 1.0, 10,and 100. At nine days post-infection, cells were washed with PBS andstained with crystal violet. One well in each 6-well dish was seededwith 293 cells to serve as a positive control for virus infection. Theresults showed that IMR90 human diploid lung fibroblasts were killeddifferentially by wild-type Ad2, dl 1010, and dl 1520. Wild-type Ad2killed the IMR90 cells effectively at an MOI of 10 or 100. Dl 1010 wasunable to replicate in IMR90 cells at the highest MOI tested, eventhough dl 1010 killed neoplastic cell lines 293, U2OS, HS700T, and DLD-1as shown above. Dl 1520 virus was able to kill IMR90 cells effectivelyonly at an MOI of 100; at this high dosage of virus the possibility thatthe cells die as a result of virus overload rather than replicationcannot be ruled out.

Thus, the data show that replication-deficient recombinant adenovirusmutants can be used to selectively kill neoplastic cells lacking p53and/or Rb function, as predicted.

Construction of Replication-Deficient Recombinant Adenovirus

The following paragraphs describe the generation and testing ofONYX-019, ONYX-020, and ONYX-021, improved adenovirus strains withdeletions in E1B 55kD which selectively replicate in p53-deficientcells. These strains have been compared to dl 1520 (hereinafter termedONYX-015) and show significant improvement over ONYX-015 in viral CPEassays.

The practice of this aspect of the invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, recombinant DNA manipulation and production, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Sambrook, MolecularCloning; A Laboratory Manual, Second Edition (1989); DNA Cloning,Volumes I and II (D. N. Glover, Ed. 1985); Oligonucleotide Synthesis (M.J. Gait, Ed. 1984); Nucleic Acid Hybridization (B. D. Hames and S. J.Higgins, Eds. 1984); Transcription and Translation (B. D. Hames and S.J. Higgins, Eds. 1984); Animal Cell Culture (R. I. Freshney, Ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide to Molecular Cloning (1984); the series, Methods in Enzymology(Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J. H.Miller and M. P. Calos, Eds. 1987, Cold Spring Harbor Laboratory),Methods in Enzymology, Volumes 154 and 155 (Wu and Grossman, and Wu,Eds., respectively), (Mayer and Walker, Eds.) (1987); ImmunochemicalMethods in Cell and Molecular Biology (Academic Press, London), Scopes,(1987); Protein Purification: Principles and Practice, Second Edition(Springer-Verlag, N.Y.); and Handbook of Experimental Immunology,Volumes I-IV (D. M. Weir and C. C. Blackwell, Eds 1986). All patents,patent applications and publications mentioned herein, both supra andinfra, are hereby incorporated by reference.

DNA constructs: Nucleotides 1339-3328 were cut out of the Ad5 pXC1plasmid obtained from Microbix Biosystems, Inc., Toronto, Ontario,Canada, using Xba1 and Bgl2 restriction enzymes. pXC1 contains Ad5sequences from base pair 22 to 5790. Nucleotides 1339-3328 encompassmost of the E1B 55kD gene; it does not include the last C-terminal 178Dase pairs. The gel purified DNA fragment was cloned into a modifiedBluscript (Stratagene Corporation) vector (pBS Bgl AHin); which replacedthe HindIII site with a Bgl2.

Oligonucleotides were made to both ends of the 1339-3328 fragment aswell as internally (see FIG. 2). The oligos are as follows:A5XBAT =5'-CCGCTCTAGAGAATGCAATAGTAGTAC-3' SEQ ID NO:1 Ad5:1339-1361ntA5BGLB =5'-CTCCAGATCTTCATGGTCATGTC-3' SEQ ID NO:2 Ad5:3315-3328ntE1B217B =5'-GGGGAATTCAAAGGCCACCCTATCCTCCGTATC-3' SEQ ID NO:3Ad5:2646-2669ntE1B275T = 5'-GGGAATTCACCGATGTAAGGGTTCGGGGCTGTG-3' SEQ IDNO:4 Ad5:2844-2868ntE1B300T = 5'-GGGAATTCTCAATTAAGAAATGCCTCTTTGAAAG-3'SEQ ID NO:5 Ad5:2919-2944ntE1B354T =5'-GGGAATTCGCCTCTCAGATGCTGACCTGCTCG-3' SEQ ID NO:6 Ad5:3081-3104nt

These oligos were used for PCR to generate different fragmentsdesignated A,B,C and D. The fragments were gel purified.

The following pairs of fragments: A and B, A and C, A and D were eachligated into the gel purified Bgl2/Xba1 7.9 Kb fragment of pXC1. Aftertransformation into DH5 alpha cells colonies were screened via PCR usingoligos: A5XBAT and A5BGLB to look for positives. See, U.S. Pat. No.5,008,182 and Hedrum, PCR Methods and Applications 2:167-71(1992).

These constructs contain DNA sequences which encode deletions of thefollowing amino acids in the E1B 55kD gene:

    ______________________________________                                        p019 (pXC1-AB)                                                                            made from fragments A and B deletion of AA                                    218-275                                                           p020 (pXC1-AC)                                                                            made from fragments A and C deletion of AA                                    218-300                                                           p021 (pXC1-AD)                                                                            made from fragments A and D deletion of AA                                    218-354                                                           ______________________________________                                    

DNA maxi preps (Qiagen) were done on one positive of each construct. TheDNA was sequenced to confirm deletions. The plasmids p019, p020 and p021are in Escherichia coli and were deposited on Dec. 20, 1996 with theAmerican Type Culture Collection, at 10801 University BuolevardManassas, Va. there are referred to as JN 019, JN 020, and JN 021,respectively. The American Type Culture Collection Accession Numbers areATCC No. 98286 for JN 019; ATCC No. 98287 for JN 020; and ATCC No. for98288 JN 021.

Virus constructs: HEK293 cells were co-transfected with the pBHGE3plasmid obtained from Microbix, and with each of the plasmids p019, p020and p021. The pBHGE3 plasmid contains a deletion of Ad5 E1 sequencesfrom base pairs 188 to 1339 and is used to construct Ad5 vectors withinsertions or mutations in the early region 1. Plaques were picked andPCR analysis was done on the first plugs using oligos A5XBAT and A5BGLBto confirm the presence of the partially deleted E1B 55kD gene. PCRmethods are well known in the art that show the use of probes in nucleicacid hybridization assays to amplify target genetic material. See forexample, U.S. Pat. Nos. 4,683,202; 4,683,195; 5,091,310; 5,008,182 and5,168,039. Plaques were expanded and viral DNA was made using the QiagenQIAamp Blood. The DNA was sequenced upstream and downstream of thedeletions to confirm the deletions.

Immunoprecipitation data: Immunoprecipitation data using virus fromlysed C33A cells showed that the viruses ONYX-019, ONYX-020 and ONYX-021(corresponding to p019, p020 and p021 above) (a) appear to make slightlymore 19kD protein than ONYX-015 using anti-19kD antibody from OncogeneSciences; (b) make a version of the 55kD protein which is of lowermolecular weight than wild-type 55kD using an anti-55kD antibody fromOncogene Sciences; note that ONYX-015 does not make detectable 55kD; and(c) reacts as expected with antibody to adenovirus using ananti-adenovirus antibody from abV Immune Response.

CPE data: Data from CPE assays using C33A comparing ONYX-015 toONYX-019, ONYX-020 and ONYX-021 showed that the new strains wereapproximately 2 to 10 fold more potent than ONYX-015 as measured by MOIrequired to produce an equivalent CPE effect.

These experiments were performed as described above. Briefly, thewild-type adenovirus Type 2 and the mutant adenoviruses, ONYX-015 toONYX-019, ONYX-020 and ONYX-021, were added to the parallel cellcultures at MOIs of 0.1, 1.0, 10, and 100. Virus suspended in PBS wasadded to the cell wells in a volume of 1 ml. The inoculated culturedishes were rocked in both the X- and Y- axes immediately afterinoculation and halfway through the adsorption time of approximately onehour at 37° C., 5% CO₂. After the one hour adsorption period, 2 ml ofDMEM with high glucose and 2% fetal bovine serum was added, and thecultures incubated at 37° C., 5% CO₂ under standard culturingconditions. Seven days post infection cell cultures were stained with0.5% crystal violet in 20% methanol in order to determine the efficacyof cell killing by the virus preparations. Dead cells were detached andrinsed out of the wells, whereas living cells remained in the well andwere stained with the dye. The results demonstrated that thereplication-deficient recombinant adenovirus preparations ONYX015,ONYX-019, ONYX-020 and ONYX-021 were able to preferentially killneoplastic cells as compared to non-neoplastic cells, and that wild-typeadenovirus Type 2 killed both neoplastic and non-neoplastic cellsapproximately equally well.

The results further demonstrated that ONYX-019, ONYX-020 and ONYX-021were 2-10 fold more potent at killing C33A cells than ONYX-015 asmeasured by a MOI required to produce an equivalent CPE effect. Theexperiment was conducted over a MOI range of 10, 1.0, 0.1, and 0.01, andONYX-019, ONYX-020 and ONYX-021 exhibited the enhanced killing at at MOIof 0.1-0.01.

Effect of Replication-Deficient Recombinant Adenovirus and Chemotherapyon Tumor Cell Killing

We next studied the efficacy of ONYX-015 (dl1520) given intravenously(IV) alone, and in combination with chemotherapy. Human tumor xenografts(colon, cervical) were grown subcutaneously in nude mice to a size of5-7 mm at which time 10⁹ total pfu were injected IV in divided doses(days 1-5). Mice in the chemotherapy groups received 5FU or cisplatinumat maximally-tolerated doses via intraperitoneal (IP) injection on day5. Controls received IV and IP vehicle injections in identical fashion.Intratumoral virus replication was noted following IV administration.While cisplatinum and 5-FU did not significantly improve survival orinhibit tumor growth, ONYX-015 alone inhibited tumor growthsignificantly (40%-60%) and improved survival (p=0.05). Combinationtherapy with ONYX-015 and either 5-FU or cisplatinum led tosignificantly improved survival versus any agent alone. Thus, ONYX-015has selective antitumoral activity when administered IV which can beimproved with chemotherapy.

Although the present invention has been described in some detail by wayof illustration for purposes of clarity of understanding, it will beapparent that certain changes and modifications may be practiced withinthe scope of the claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 6                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #             27   AATA GTAGTAC                                               - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #                23TCAT GTC                                                   - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #         33       ACCC TATCCTCCGT ATC                                        - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #         33       AAGG GTTCGGGGCT GTG                                        - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #        34        GAAA TGCCTCTTTG AAAG                                       - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #          32      GATG CTGACCTGCT CG                                         __________________________________________________________________________

What is claimed is:
 1. A recombinant plasmid, JN019, on deposit with theAmerican Type Culture Collection with Accession number
 98286. 2. Arecombinant plasmid, JN020, on deposit with the American Type CultureCollection with Accession number
 98287. 3. A recombinant plasmid, JN021,on deposit with the American Type Culture Collection with Accessionnumber
 98288. 4. An isolated nucleic acid sequence present in arecombinant plasmid, JN019, said plasmid on deposit with the ATCC withaccession number 98286, said nucleic sequence having a deletion in theE1B region of adenovirus, said deletion encoding amino acids 218-275. 5.An isolated nucleic acid sequence present in a recombinant plasmid,JN020, said plasmid on deposit with the ATCC with accession number98287, said nucleic sequence having a deletion in the E1B region ofadenovirus, said deletion encoding amino acids 218-300.
 6. An isolatednucleic acid sequence present in a recombinant plasmid, JN021, saidplasmid on deposit with the ATCC with accession number 98288, saidnucleic sequence having a deletion in the E1B region of adenovirus, saiddeletion encoding amino acids 218-354.
 7. A host cell comprising aplasmid selected from the group consisting of JN019, JN020, and JN021.8. The replication deficient adenovirus Onyx
 019. 9. The replicationdeficient adenovirus Onyx
 020. 10. The replication deficient adenovirusOnyx 021.